Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV

Abstract

The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics and competition surface plasmon resonance (SPR) analyses suggested that this compound acts as a competitive inhibitor with an IC50 of 6 μM against MERS-CoV PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-CoV PLpro with an IC50 of 11 μM. These results raised the possibility that inhibitor recognition specificity of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition, inhibitory activity of this compound was selective for SARS-CoV and MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).

Figure 1

Schematics of SARS-CoV and MERS-CoV polyproteins. (A) Cleavage positions of PLpro (pink) and 3CLpro (cyan) are shown by different colored arrows in their polyproteins. (B) Cleavage site comparison between SARS and MERS PLpro enzymes. Sequence motifs recognized by SARS-CoV PLpro (SARS-PLpro) and MERS-CoV PLpro (MERS-PLpro) are LXGG↓(A/K)X and (L/I)XGG↓(A/D)X, respectively.

Figure 2

SARS-PLpro lead inhibitors and structures. (A) Structures of four SARS-PLpro lead inhibitors (I-1–I-4).^, (B) X-ray crystal structure of inhibitor I-2 bound to SARS-PLpro (PDB: 3E9S). The amide group of inhibitor I-2 forms two hydrogen bonds with D165 and Q270 in the BL2 loop. (C) X-ray crystal structure of inhibitor I-3 bound to SARS-PLpro (PDB: 3MJ5). The amide group of inhibitor I-3 forms a hydrogen bond with Q270 in the BL2 loop. The aromatic ring of Y269 forms a hydrophobic interaction with the naphthyl rings of both I-2 and I-3. The three catalytic site residues are shown in green. (D) Overlay of the SARS-PLpro blocking loop 2 (BL2) and the corresponding loop of MERS-PLpro.

Figure 3

Structure comparison of SARS-PLpro complexes and MERS-PLpro. (A) Overlay of five SARS-PLpro complex structures with an inhibitor or a substrate, apo SARS-PLpro, apo MERS-PLpro, and MERS-CoV-PLpro complex with a ubiquitin. The PDB codes of aligned structures are Apo MERS-CoV-PLpro (PDB: 4RNA), MERS-CoV-PLpro complex with a ubiquitin (PDB: 4RF1), Apo SARS-CoV-PLpro (PDB: 2FE8), SARS-CoV-PLpro inhibitor complex with inhibitor I-2 (PDB: 3E9S), SARS-CoV-PLpro complex with inhibitor I-3 (PDB: 3MJ5), SARS-CoV-PLpro complex with inhibitor 3k (PDB: 4OVZ), SARS-CoV-PLpro complex with inhibitor 3j (PDB: 4OW0), and SARS-CoV-PLpro complex with ubiquitin aldehyde substrate (PDB: 4MM3). (B) Expanded overlaid structures of BL2 and surrounding residues involved with inhibitor binding. (C) Different orientation of Figure 4B, showing catalytic residues and relative BL2 orientations. (D) Expanded overlaid structures of BL2 loops and a ubiquitin aldehyde (blue) and ubiquitin (orange) substrates for SARS-PLpro and MERS-PLpro, respectively. Ubiquitin is hidden, and only part of the each substrate is shown in this figure due to space constraints. (E) The active site, catalytic triad (CT), and two blocking loop (BL1 and BL2) residues of MERS-PLpro and their corresponding aligned residues in the active sites of SARS-PLpro and three bat coronaviral PLpro enzymes.

Figure 4

HTS results from Life Chemicals antimicrobial/antiviral focused library and hit validation. (A) Schematic of HTS with 25 000 Life Chemicals compounds and hit validation process. (B) Bar graphs of IC₅₀ values and the dissociation equilibrium constants (K_D) of six hit compounds determined by fluorescence-based enzymatic assay and Surface Plasmon Resonance (SPR), respectively. All data were normalized for immobilization levels of target proteins and reference. Bars that reach the top of the graph represent either IC₅₀ or K_D values of over 200 μM (no inhibition or no binding).

Figure 5

Mechanism of inhibition. Dixon plots of compound 4 against SARS-PLpro (A) and MERS-PLpro (B). (C) Summary table of kinetic mode of inhibition of compound 4. Mechanism of enzyme inhibition of compound 4 was determined to be a mixed inhibition for SARS-PLpro and a competitive inhibition for MERS-PLpro. Determined K_i values of compound 4 were 11.5 μM and 7.5 μM for SARS-PLpro and MERS-PLpro, respectively. (D) IC₅₀ value comparison of four SARS-PLpro lead inhibitors in combination with the newly identified compound 4 to determine if they inhibit synergistically. (E) Bar graphs of the dissociation equilibrium constants (K_D) of compound 4 in the absence (solid bars) and in the presence (striped bars) of substrate determined by Surface Plasmon Resonance (SPR).

Figure 6

Selectivity of compound 4. (A) Structural alignment of MERS-PLpro with two human deubiquitinating enzymes. The aligned catalytic triads of two human ubiquitin C-terminal hydrolases, hUCH-L1 (green, PDB: 2ETL) and hUCH-L3 (orange, PDB: 1UCH), are shown with that of MERS-PLpro (tan, PDB: 4RNA) in the expanded box. (B) Selectivity of the confirmed hit compound 4. In addition to two human cysteine proteases (hUCH-L1 and hUCH-L3), two unrelated enzymes, Hepatitis C Virus NS3 serine protease (NS3) and Bacillus anthracis dihydroorotase (PyrC), were also tested along with both PLpro enzymes.

Figure 7

Active site analysis of the MERS-PLpro. (A) Active site alignment of MERS-PLpro (tan) and SARS-PLpro (cyan). The three catalytic triad residues (C111, H278, and D293) of MERS-PLpro are aligned with the SARS-PLpro catalytic triad (C112, H273 and D287). (B) Sequence alignment of important residues near the catalytic triad between various CoV. Residue numbers are shown for MERS-PLpro. (C) Potential mechanism 1 for oxyanion hole stabilization via N109. Active site and substrate residues are shown in green and pink, respectively. (D) Potential mechanism 2 for oxyanion hole stabilization via N109. (E) Enzyme activity comparison between wild-type and two mutant MERS-PLpro enzymes.